We have determined that certain proteins, including polypeptides with Mr's of 30,000, 35,000, 24,000, and 20,000, are expressed in low- passage, virulent strains of Borrelia burgdorferi (Bb) but are absent or underexpressed in high-passage, avirulent strains. The central hypothesis of this proposal is that these 'virulence-associated proteins' are important virulence determinants and immune targets in Bb and thus will be useful in the diagnosis, epidemiology, and possible prevention of Lyme disease. In Specific Aim 1, we will identify virulence-associated proteins of Bb by comparing the protein profiles of virulent and avirulent isogeneic variants using non-equilibrium pH gradient two dimensional gel electrophoresis (2DGE), which separates the polypeptides of Bb into a series of distinct, easily differentiated spots. The protein profiles of low-passage North American, European, and Asian strains will also be compared to determine the degree of variation and the correlation, if any, between expression of certain proteins and pathogenic properties such as a propensity for arthritogenesis. Specific Aim 2 will consist of the biochemical and genetic characterization of selected virulence-associated proteins, and will include determination of partial amino acid sequence, gene sequence, gene location (i.e.plasmid-encoder vs. chromosomal) and structural properties. In preliminary studies, we have cloned the gene of a major, 30K, non-OspA, virulence-associated protein of the B31 strain of Bb. Analogous genes in Bb strains from different geographical locations will be examined to determine the degree of heterogeneity among Bb isolates. In Specific Aim 3, the immune response of Lyme disease patients and Bb-infected C3H/HeN mice to the 30 kDa protein and other virulence-associated proteins will be examined to determine their utility for immunodiagnosis and immunoprotection. Antibody responses to these proteins will be determined using 2DGE Western blot reactivity and (as recombinant antigens become available) ELISA assays, testing both IgM and IgG reactivity. The possible immunoprotective activity of the virulence-associated proteins will be examined by active immunization of C3H/HeN mice with the 30K protein or other proteins and by passive protection studies with monoclonal or monospecific antibodies. This project is likely to provide exciting new information regarding these previously uncharacterized and potentially important Bb proteins, their role in pathogenesis, and their utility in Lyme disease diagnosis and prevention.